The effects of oats on the function of gut microflora in children with coeliac disease

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1 The effects of oats on the function of gut microflora in children with coeliac disease B. Tjellstrom, Lars Stenhammar, Tommy Sundqvist, Karin Fälth-Magnusson, Elisabet Hollén, Karl-Eric Magnusson, E. Norin, T. Midtvedt and Lotta Högberg Linköping University Post Print N.B.: When citing this work, cite the original article. Original Publication: B. Tjellstrom, Lars Stenhammar, Tommy Sundqvist, Karin Fälth-Magnusson, Elisabet Hollén, Karl-Eric Magnusson, E. Norin, T. Midtvedt and Lotta Högberg, The effects of oats on the function of gut microflora in children with coeliac disease, 2014, Alimentary Pharmacology and Therapeutics, (39), 10, Copyright: Wiley Postprint available at: Linköping University Electronic Press

2 1 February 25th, 2014 The effects of oats on the function of gut microflora in children with coeliac disease Tjellström B*,#, Stenhammar L #,, Sundqvist T^, Fälth-Magnusson K,, Hollén E^, Magnusson K-E^, Norin E *, Midtvedt T *, Högberg L #, * Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, Stockholm, Sweden # Department of Paediatrics, Norrköping Hospital, County Council of Östergötland, Norrköping, Sweden Division of Paediatrics, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden ^ Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Faculty of Health Sciences, Linköping University, Linköping, Sweden Department of Paediatrics, Linköping University Hospital, Linköping, County Council of Östergötland, Sweden Corresponding author: Bo Tjellström MD PhD bo.tjellstrom@gmail.com Department of Microbiology, Tumor and Cell Biology Karolinska Institute Nobels väg 16 SE Stockholm Phone Running title: Effect of oats on gut microflora in coeliac disease

3 2 Keywords: Coeliac disease; gluten-free diet; oats; faecal short chain fatty acids; gut microflora. Word count: 2,052 (excluding title page, references and tables)

4 3 SUMMARY Background Faecal short chain fatty acids (SCFAs) are produced by the gut microflora. We have previously reported high faecal SCFA levels in children with CD, indicating alteration in gut microfloral metabolism. Data accumulated over recent decades by us and others suggest that wheat-free oats can safely be included in a gluten-free diet (GFD). However, concerns have been raised with respect to the safety of oats in a subset of coeliacs. Aim To describe faecal SCFA patterns in children with newly diagnosed CD treated for one year with a GFD with or without oats. Methods This report is part of a randomised, double-blind study on the effect of a GFD containing oats (GFD-oats) versus a standard GFD (GFD-std). Faecal samples were received from 34 children in the GFD-oats group and 37 in the GFD-std group at initial diagnosis and/or after one year on a GFD. Faecal SCFAs were analysed. Results The GFD-std group had a significantly lower total faecal SCFA concentration at 12 months compared with 0 months (p<0.05). In contrast, total SCFA in the GFD-oats group remained high after one year on the GFD. The children in the GFD-oats group had significantly higher acetic acid (p<0.05), n-butyric acid (p<0.05) and total SCFA concentration (p<0.01) after one year s diet treatment compared to the GFD-std group. Conclusions

5 4 Our results indicate that oats do affect the gut microflora function, and that some coeliac children receiving oats may develop gut mucosal inflammation, that may present a risk for future complications.

6 5 INTRODUCTION Coeliac disease (CD) is a gluten-induced, immune-mediated, inflammatory process affecting almost exclusively individuals carrying HLA DQ2 and/or DQ It is characterised by increased coeliac serology markers, and small bowel enteropathy including lymphocyte infiltration of the epithelium and lamina propria, crypt hyperplasia and various grades of villous flattening. Treatment with a gluten-free diet (GFD), i.e. a diet free of wheat, rye and barley, leads to a normalisation of serology markers and enteropathy. The prevalence of CD is about 1 % in the Western World. 4 CD is one of the most common chronic diseases in the childhood population, with a prevalence as high as 3 %. 5 There is mounting evidence that gut microorganisms trigger immune activation in CD 6 7, and aberrations in the gut microflora have been reported in coeliacs Furthermore, our group has analysed faecal short chain fatty acid (SCFA) concentration as a marker of gut microflora metabolism. We found that children with untreated, symptomatic as well as asymptomatic CD, and coeliac children treated with GFD for up to one year have SCFA patterns that differ significantly from those seen in healthy controls, indicating a disturbed microbiota in CD Oats have been allowed in the GFD in order to increase dietary treatment compliance in these children. The first randomised study on diets with and without oats in adults with CD was published by Janatuinen 14 in 1995 and the first randomised, double blind study on the effect of oats in a large paediatric CD population was performed by our group. 15 Although these studies indicated that wheat-free oats could safely be included in the GFD of coeliac adults and children, concerns have been raised that it may not be fully safe in a subset of coeliacs. Thus, the aim of the present study was to address this issue by examining the faecal SCFA

7 6 pattern in CD children after one year s treatment with or without oats in their GFD to see if oats affect the gut microflora. The children studied comprised two sub-groups of subjects included in the previous study by our group. 15 MATERIAL AND METHODS Patients This study was part of a randomised, double blind, multicentre study on the effect of a GFD containing oats (GFD-oats) compared to a standard GFD (GFD-std) in 116 children (GFD oats, n=57; GFD std, n=59) with newly diagnosed CD, and has previously been described in detail. 15 The oats were strictly wheat-free. We aimed at a daily oats intake of 25 50g. Thirtyfour children from the GFD-oats group and 37 children from the GFD-std group were included in this sub-study. Table 1 presents details of the children. The study included all children who delivered faecal samples large enough for SCFA analysis. Each child was studied over a period of one year and delivered at least one faecal sample at 0, 3, 6 and/or 12 months. At the start of the study, all children had small bowel enteropathy consistent with CD, without any difference in the level of mucosal damage between the study groups. At 12 months, all children were in clinical remission and all showed a normalisation of their intestinal mucosa, except for one child in the GFD-std group, who did not undergo a control biopsy. Gut microflora-associated characteristics A microflora-associated characteristic (MAC) is defined as the recording of any anatomical structure, physiological, biochemical or immunological function in an organism that has been influenced by the microflora. 16 In the present study we used the faecal SCFA pattern as MAC, in order to assess the effects of oats in our CD children. The SCFA patterns found in the

8 7 faecal samples represent the unabsorbed fraction of SCFAs produced in the gastrointestinal tract. The faecal samples were frozen either immediately or at least within 20 min of passage and stored at -20 o C pending analysis. SCFA analyses were performed at the Karolinska Institute, Stockholm, Sweden. The faecal material was homogenised after the addition of distilled water containing 3 mmol/l of 2-ethylbutyric acid (=internal standard) and H 2 SO 4 (0.5 mmol/l). A 2-mL sample of the homogenate was vacuum distilled according to the method of Zijlstra et al., 17 modified by Höverstad & Björneklett. 18 The distillate was analysed using gas-liquid chromatography and quantified using internal standardisation. Flame ionization detection was employed. The results were expressed in mmol/kg wet weight. The following SCFAs were analysed: acetic acid, propionic acid, i-butyric acid, n-butyric acid, i-valeric acid and n-valeric acid. The results are presented as (i) individual and total SCFA concentrations, and (ii) a fermentation index (amount of acetic acid minus propionic acid and n-butyric acid, together divided by the total amount of SCFAs) This index mainly reflects the fermentation of carbohydrates, and a high value combined with a high total amount of SCFA principally mirrors pro-inflammatory properties of the SCFAs. Statistical analysis Statistical analysis was performed using Student s t-test. P-values <0.05 (*) and < 0.01 (**) were considered significant. Ethical considerations The oats study was approved by the Human Research Ethics Committee of the Faculty of Health Sciences, University of Linköping, as main committee for the investigation.

9 8 RESULTS In the GFD-std group, the total SCFA concentration was high at 0 and 6 months but significantly lower after 12 months on GFD (p<0.05) (Table 2). In contrast, the total SCFA remained at a high level throughout the year on a diet in the GFD-oats group. The children in the GFD-oats group had significantly higher acetic acid (p<0.05), n-butyric acid (p<0.05) and total SCFA (p<0.01) concentrations after one year of diet treatment compared to the GFD-std group. The concentrations of the remaining SCFAs did not differ between the study groups at 0, 6 and 12 months, respectively. During the year on a GFD, the fermentation index remained high in both the GFD-oats and GFD-std groups with no significant differences. Mean (median) amount of oats ingested in the GFD-oats group was 20 (15) and 16 (15) g/day at 6 and 12 months, respectively (no significant difference). We did not find any conclusive correlation between the amount of consumed oats and SCFA levels. The number of faecal samples at 3 and 9 months were too few to permit statistical analysis. Results of these samples were thus excluded from the analyses. DISCUSSION The main finding in the present study was the significant decrease in total SCFA concentration in the GFD-std group during the year on a GFD. This is in contrast to the GFDoats group, in which the pro-inflammatory acetic acid and total SCFA concentration remained high throughout the diet period. This might be explained by the higher fibre content, due to the addition of oats to the GFD, thus providing more substrate for fermentation. However, it has been shown that poorly-fermentable oat husk does not result in an increase in production

10 9 of total SCFA, acetic acid, propionic acid or butyric acid, as opposed, for example, to the highly-fermentable soy-bean oligo-saccharide. 21 Alternatively, it could have been that a number of children in the GFD-oats group did not tolerate oats and that the high faecal SCFA concentration in the GFD-oats group was indicative of an on-going inflammation in the oatsexposed intestinal mucosa. Although acetic acid is generally regarded as a fatty acid that can promote an inflammatory process, recent evidence from animal experiments suggests that it could also play an immune-modulating or dual role through its effects on chemoattractant receptors on neutrophils, potentially adding to a more rapid activation and resolution of the inflammatory reaction. The SCFA fermentation index, which mirrors intestinal inflammation and has a reference value of < 0.05, was high in both groups after one year s GFD. We recently reported that a GFD-std of more than one year s (median 4 years ) duration is needed to fully normalise faecal SCFA patterns, including the fermentation index, in children with CD. 20 The present paper is based on the results from one year s diet therapy, only. It thus remains to be seen if normalisation of SCFA levels and fermentation index in the GFD-oats group occurs following longer periods on a GFD. Given that oats may have the potential to elicit an inflammatory response in some patients with CD, it is important to maintain long-term monitoring of CD patients receiving oats, considering the fact that chronic gut mucosal inflammation might increase the risk of malignancy, which is a potential, even though small, risk factor in CD, documented in a recent meta-analysis. 24 In our original study on the effects of oats in children with newly diagnosed CD, we concluded that the addition of oats to a GFD was accepted and tolerated by the majority of children studied, as indicated by normalisation of the small bowel mucosal architecture and

11 10 decreasing coeliac serology markers after one year s treatment with a GFD containing oats. 15 Similar results were reported by others On the other hand, Lundin et al. raised some concerns regarding the safety of oats in adults with CD They concluded that a number of adult coeliacs exposed to oats in their GFD appear to experience abdominal symptoms or even produce avenin-reactive T-cells in the small intestinal mucosa. However, Koskinen et al. reported that oats did not induce transglutaminase-2 autoantibody production at the intestinal mucosal level in CD children over a 2-year follow-up. 25 Furthermore, we have previously reported that CD children have similar serum anti-avenin antibody titres following one year s GFD-std or GFD-oats treatment. 29 Nevertheless, it should be noted that some of the CD children in both study groups continued to excrete high amounts of urinary nitrite/nitrate, which is an indication of up-regulation of inducible nitric oxide synthase, induced by proinflammatory cytokines. 30 Moreover, it has been shown that the potential toxic effects of oats in coeliacs may differ between the various varieties of oats Further studies of the longterm effects of different oats varieties in coeliac patients are thus warranted, 33 since oats have a positive nutritional effect in these patients significantly increasing the fibre content of the conventional GFD, the fibre quantity of which is otherwise poor. 26 Importantly, the oats in this study were strictly wheat-free, a necessary demand also in future studies. A limitation of this randomised, double-blind study is the fact that all children in the study groups did not deliver faecal samples. In addition, some of the delivered samples were too small to permit analysis. However, the distribution of missing samples is evenly distributed between the two study groups. Moreover, it can be questioned whether measurement of faecal SCFA at just 1 2 time points over several months is meaningful, given the likely variability of daily food intake and volume of faeces. However, in our experience, the functional aspects and the relative proportion of faecal SCFAs appear to vary more slowly, allowing for less

12 11 frequent sampling and thus giving relevance to our results. This is also supported by previous reports by us. In this study we focused our attention on elucidating oats-related effects on the function of the gut microflora of the children. Based on the new findings, it would also be of interest to study if there are corresponding, distinct traits in the composition of the faecal microbiota. In conclusion, the results of this study indicate that oats in the GFD of children with CD affect the faecal SCFA pattern, a gut microflora-associated characteristic. To our knowledge, this observation has not previously been reported. The clinical implication of this finding is unknown. However, some coeliac children receiving oats may suffer chronic gut mucosal inflammation that presents a potential risk for future complications. Obviously, long-term studies and new bio-markers of gut mucosal inflammation are needed if we are to further analyse the effects of oats in patients with CD. AUTHORSHIP Guarantor of the article: BT Author contributions: LH (principal investigator) and LS designed the original oats study and performed it together with K F-M. BT, EN and TM were responsible for the faecal analyses and TS for the acquisition of data and statistical analysis. K-E M contributed with expert knowledge in the field. EH had an important role as co-ordinator of the research material and compared it with previous analyses of urine and blood samples from the study patients. All authors made substantial contributions to the drafting of the manuscript, revisited it critically and approved the final version.

13 12 ACKNOWLEDGEMENT We thank Associate Professor Gunnar Jansson, Dr. Jan-Åke Hammersjö and Dr. Urban Myrdal for their contribution of material in this study. We also thank Anna-Karin Persson for excellent work with the faecal sample analyses. The study was financially supported by the Swedish Medical Research Council. DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. REFERENCES 1. Di Sabatino A, Corazza GR. Coeliac disease. Lancet 2009;373: Husby S, Koletzko S, Korponay-Szabi IR, et al. European Society for Pediatric Gastroenterology Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 2012;54: Ludvigsson JF, Leffler DA, Bai JC, et al. The Oslo definitions for coeliac disease and related terms. Gut 2013;62: Kang JY, Kang AHY, Green A, et al. Systematic review: worldwide variation in the frequency of coeliac disease and changes over time. Aliment Pharmacol Ther 2013;38: MyléusA, Ivarsson A, Webb C, et al. Celiac disease revealed in 3 % of Swedish 12- year-olds born during an epidemic. J Pediatr Gastroenteol Nutr 2009;49: Meresse B, Malamut G, Cerf-Bensussan N. Celiac disease: an immunological jigsaw. Immunity 2012;36: Sollid LM, Jabri B. Triggers and drivers of autoimmunity: lessons from coeliac disease. Nat Rev Immunol 2013;13: Ou G, Hedberg M, Hörstedt P, et al. Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease. Am J Gastroenterol 2009;104:

14 13 9. Di Cagno R, Rizzello CG, Gagliardi F, et al. Different fecal microbiotas and volatile organic compounds in treated and untreated children with celiac disease. Appl Environ Microbiol 2009;75: Sanz YS, De Palma G, Laparra M. Unravelling the ties between celiac disease and intestinal microbiota. Int Rev Immunol 2011;30: Nistal E, Caminero A, Herrán AR, et al. Differences of small intestinal bacteria populations in adults and children with/without celiac disease: effect of age, gluten diet, and disease. Inflamm Bowel Dis 2012;18: Tjellström B, Stenhammar L, Högberg L, et al. Gut microflora associated characteristics in children with celiac disease. Am J Gastroenterol 2005;100: Tjellström B, Stenhammar L, Högberg L, et al. Screening-detected and symptomatic untreated celiac children show similar gut microflora-associated characteristics. Scand J Gastroenterol 2010;45: Janatuinen EK, Pikkarainen PH, Kemppainen TA, et al. A comparison of diets with and without oats in adults with celiac disease. N Engl Med 1995;333: Högberg L, Laurin P, Fälth-Magnusson K, et al. Oats to children with newly diagnosed coeliac disease: a randomised double blind study. Gut 2004;53: Midtvedt T, Björneklett A, Carlstedt-Duke B, et al. The influence of antibiotics upon microflora-associated characteristics in man and mammals. In: Wostmann BS et al., eds. Germ-free research, microflora control and its application to the biomedical sciences. Progress in clinical and biological research, vol New York: Alan R. Liss Corporation Inc.;1985, pp Zijlstra JB, Beukema J, Wolthers BG, et al. Pretreatment methods prior to gaschromatographic analysis of volatile fatty acids from faecal samples. Clin Chim Acta 1977;78: Höverstad T, Björneklett A. Short-chain fatty acids and bowel functions in man. Scand J Gastroenterol 1984;19: Tjellström B, Högberg L, Stenhammar L, et al. Effect of exclusive enteral nutrition on gut microflora function in children with Crohn s disease. Scand J Gastroenterol 2012;47: Tjellström B, Högberg L, Stenhammar L, et al. Faecal short chain fatty acid pattern in childhood coeliac disease is normalised after more than one year s gluten-free diet. Microb Ecol Health Dis 2013;24: DOI: /mehd.v24i Kapadia SA, Rimundo AH, Grimble GK, et al. Influence of three different fibersupplemented enteral diets on bowel function and short-chain fatty acid production. J Parenter Enteral Nutr 1995;19:63-8.

15 Maslowski KM, Vieira AT, Ng A et al. Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43. Nature 2009;461: Maslowski KM, Mackay CR. Diet, gut microbiota and immune responses. Nat Immunol 2011;12: Tio M, Cox MR, Eslick GD. Meta-analysis: coeliac disease and the risk of all-cause mortality, any malignancy and lymphoid malignancy. Aliment Pharmacol Ther 2012;35: Koskinen O, Villanen M, Korponay-Szabo I, et al. Oats do not induce systemic or mucosal autoantibody response in children with coeliac disease. J Pediatr Gastroenterol Nutr 2009;48: Fric P, Gabrovska D, Nevoral J. Celiac disease, gluten-free diet, and oats. Nutr Rev 2011;69: Lundin KEA, Nilsen EM, Scott HG, et al. Oats induced villous atrophy in coeliac disease. Gut 2003;52: Arentz-Hansen H, Fleckenstein B, Molberg O et al. The molecular basis for oat intolerance in patients with celiac disease. PLoS Med 2004;1: Hollén E, Holmgren Peterson K, Sundqvist T, et al. Coeliac children on a glutenfree diet with or without oats display equal anti-avenin antibody titres. Scand J Gastroenterol 2006;41: Hollén E, Forslund T, Högberg L, et al. Urinary nitric oxide during one year of gluten-free diet with or without oats in children with coeliac disease. Scand J Gastroenterol 2006;41: Silano M, Dessi M, de Vincenzi M, et al. In vitro tests indicate that certain varieties of oats may be harmful to patients with celiac disease. J Gastroenterol Hepatol 2007;22: Silano M, Di Benedetto R, Maialetti, F et al. Avenins from different cultivars of oats elicit response by celiac peripheral lymphocytes. Scand J Gastroenterol 2007;42: Troncone R, Ivarsson A, Szajewska H, et al. Review article: future research on coeliac disease a position report from the European multistakeholder platform on coeliac disease (CDEUSSA). Aliment Pharmacol Ther 2008;27:

16 15 Table 1. Study data. Group GFD-oats GFD-std No. of children Age (yrs) (mean) (median) (range) Males/females 11/23 12/25 GFD-oats=gluten-free diet including oats GFD-std =standard gluten-free diet

17 16 Table 2. Results of gut microbial activity, as described by short chain fatty acid (SCFA) levels and SCFA Fermentation Index (see text), in children treated with a gluten-free diet (GFD) with or without oats for one year. Group GFD-oats GFD-std No. of children GFD diet (months) SCFA Acetic acid * Propionic acid i-butyric acid n-butyric acid * i-valeric acid n-valeric acid Total SCFAs ** Fermentation Index GFD-oats=gluten-free diet including oats; GFD-std=standard gluten-free diet; SCFA=short chain fatty acid. Mean (mmol/kg faeces) + SEM. Significant difference vs. before diet (0 months) (p<0.05). * ; **Significant difference vs. GFD-std 12 months (*p<0.05;**p<0.01).

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The effects of oats on the function of gut microflora in children with coeliac disease

Alimentary Pharmacology and Therapeutics The effects of oats on the function of gut microflora in children with coeliac disease B. Tjellstr om*,, L. Stenhammar,, T. Sundqvist, K. F alth-magnusson,, E.